Transcobalamin II assay method

ABSTRACT

An assay method for determining transcobalamin saturation wherein a transcobalamin containing liquid sample is contacted with a porous substrate with immobilized thereon a transcobalamin immobilizing ligand and with a reporter-labelled transcobalamin binding partner and wherein signals from reporter labels which become immobilized on said substrate are detected, characterised in that one of said ligand or said binding partner comprises a first ligand or binding partner capable of specific binding to holo transcobalamin and a second ligand or binding partner capable of binding to apo transcobalamin or to holo and apo transcobalamin.

CROSS REFERENCES TO THE RELATED APPLICATIONS

This application is a divisional application of pending of U.S. patentapplication Ser. No. 10/474,506, filed Mar. 4, 2004, now allowed (ofwhich the entire disclosure of the pending, prior application is herebyincorporated by reference).

The invention relates to improvements in and relating to diagnosticassay methods, in particular assays for transcobalamin.

Cobalamin or vitamin B₁₂ is a water-soluble vitamin which forms part ofthe vitamin B complex found in foods. The core molecule consists of acorrin ring of four pyrole units which surround the essential cobaltatom. Cobalamin is the only vitamin which cannot be synthesised byanimals or plants and must be absorbed from food in the gut. It canhowever be stored in the liver. It is synthesised by micro-organisms, inparticular by anaerobic bacteria and yeasts.

Cobalamin functions in vivo as a co-enzyme and cobalamin enzymescatalyse three types of reaction: (i) intra-molecular rearrangements,for example, the formation of succinyl CoA from L-methylmalonyl CoA;(ii) methylations, for example, the formation of methionine bymethylation of homocysteine; and (iii) reduction of ribonucleotides todeoxyribonucleotides in some micro-organisms. In mammals, only twoenzymic reactions, those specifically mentioned in (i) and (ii) above,are known to require cobalamin as a co-enzyme.

In the process of digestion, a salivary protein called haptocorrin,hereinafter referred to as HC (which is also referred to in the art asR-binder or transcobalamins I and III collectively), binds cobalamin inthe upper gastrointestinal tract forming a complex which passes throughthe stomach. Pancreatic enzymes digest the cobalamin-haptocorrin(holo-HC) complex in the ileum, liberating cobalamin which is then boundto a protein called intrinsic factor, which is secreted by the gastricmucosa, to form a further complex. The cobalamin-intrinsic factorcomplex binds to a specific receptor in the lining of the terminalileum, whereupon it is dissociated by a releasing factor and thecobalamin transported actively across the membrane of the ileum into theblood stream.

Cobalamin does not circulate in the body in a free form in anappreciable amount. Probably 99% or so of cobalamin is bound byhaptocorrin, transcobalamin or albumin.

The protein believed to be responsible for transporting cobalamin totarget tissues is transcobalamin II (hereinafter simply referred to astranscobalamin or TC), a critical trace protein without which cobalamincannot cross cell membranes. Despite this important metabolic function,only about 6-25% of cobalamin in the serum is bound to TC and most iscarried by HC. TC is a single chain polypeptide of 45 kDa foundprimarily in serum, seminal fluid and cerebro-spinal fluid. Cobalaminbound TC or holo-TC, attaches to specific receptors on cell membranesand once bound, the holo-TC complex is taken into cells by pinocytosis.

TC is synthesised by the liver, vascular endothelium, enterocytes,macrophages and fibroblasts and circulates predominantly as apo-TC, i.e.lacking bound cobalamin. It has a short half life of approximately 90minutes.

Less than a quarter of the total plasma cobalamin is associated with TC.The rest is bound to HC or albumin as mentioned above.

Since cobalamin must be absorbed from food, any conditions which resultin impaired gastric function, for example, gastroenteritis or conditionsresulting in gastric atrophy, or an inability to produce functionalhaptocorrin, intrinsic factor, releasing factor, TC or TC receptors, canresult in impaired uptake of cobalamin and resultant deficiency.

Certain population sub-groups, for example the aged, pregnant women,patients with chronic or acute gastrointestinal disease, those sufferingfrom certain autoimmune diseases, those with a family history ofpernicious anaemia and AIDS sufferers, are particularly prone tocobalamin deficiency.

The clinical manifestations of cobalamin deficiency are varied andnumerous but primarily involve anaemia, megaloblastic haematopoiesis andfunctional and structural disorders of the nervous system. Around 60% ofindividuals diagnosed as being deficient in cobalamin are anaemic, butin many neurological symptoms are the only clinical signs observed.Around 10% of patients exhibit psychiatric symptoms and around 40%exhibit both neurological and psychiatric symptoms.

Early diagnosis of cobalamin deficiency is crucial to ensure a goodprognosis for patients, since some of the manifestations of cobalamindeficiency, particularly the neuropsychiatric effects, are irreversibleif not detected and alleviated by cobalamin therapy quickly.

It is desirable therefore to accurately assess the cobalamin level of anindividual in an expedient and efficient manner, with a view toestablishing whether or not the individual may be suffering fromcobalamin deficiency.

Measurement of total plasma cobalamin, i.e. cobalamin (and cobalaminlike substances) bound to HC or TC, has been used in attempts to assesscobalamin deficiency. This technique results in a broad basedconcentration distribution within a population which is considered to benormal and hence produces a wide reference range. Within individualshowever, the range of available cobalamin considered to be normal forthat individual, is very narrow. It has been observed that although anindividual's metabolically active cobalamin concentration has movedoutside their own reference range, their total plasma cobalamin contentremains within the range considered to be normal for the population.Under such circumstances, cobalamin deficiency can go undetected. Suchan unreliable method is clearly undesirable and it is well recognisedthat such serum or plasma cobalamin measurements have low diagnosticsensitivity and specificity.

Microbial assays involving micro-organisms dependent upon cobalamin forgrowth, have been developed and used in measuring plasma cobalaminconcentration, but in addition to the difficulty of estimating theappropriate reference range, these methods require extraction andconversion of the cobalamins which is very time consuming, troublesomeand wholly unsuited for rapid laboratory screening.

Alternative methods for assessing cobalamin deficiency involve measuringthe accumulation of metabolites in the plasma which require cobalaminfor their conversion. Plasma methylmalonate and plasma homocysteinelevels increase in cobalamin deficient individuals and make goodcandidate molecules for correlation with vitamin B₁₂ deficiency. Methodsbased on homocysteine assessment have been shown, however, to becomplicated, impractical and show poor specificity and sensitivity.Whilst methods based on methylmalonate measurement are accurate andreliable, they are cumbersome and require analysis by combinedgas-chromatography/mass-spectrometry and are hence expensive and againunsuitable for routine clinical screening.

It has also been suggested that measurement of TC bound cobalamin asopposed to total plasma cobalamin may provide a reliable clinicalindictor of the likelihood of cobalamin deficiency (Herbert et al.(1990) Am. J. Hematol. 34:132-139; Wickramasinghe and Fida (1993) J.Clin. Pathol. 46:537-539; U.S. Pat. No. 4,680,273). Such efforts todetermine holo-TC concentration were mostly indirect, estimating holo-TCconcentration as the difference between total plasma cobalamin and thecobalamin concentration of TC depleted plasma.

Such TC depletion may be accomplished by adsorption to ammonium sulphate(Carmel (1974) Am. J. Clin. Pathol. 62:367-372), microsilica (Herzlich &Hubert (1988) Lab. Invest. 58:332-337; Wickramasinghe & Fida (1993) J.Clin. Pathol. 46:537-539), microfine glass (Vu et al. (1993) Am. J.Hematol. 42:202-211) or immobilized anti-TC polyclonal antibodies(Lindemans et al. (1983) Clin. Chim. Acta 132:53-61). The concentrationof cobalamin in total plasma and the depleted fraction is performed bymethods well known in the art such as radio or enzyme immunoassaytechniques. These methods are unsuitable for routine screening whetherautomated or not automated because they are complex and time consumingand because the low degree of specificity of the adsorptive materialsused results in insufficient separation of holo-TC and holo-HC resultingin an overestimation of holo-TC. Lot-to-lot variation of the adsorptivematerial introduces further errors and most importantly, the subtractionof one large volume from another large volume results in unacceptableinaccuracies and unreliability.

Other attempts to assess TC have involved separating TC from other serumcomponents, including HC, using its lipophilicity. Thus Kapel et al.(1988) Clin. Chim. Acta 172:297-310, Benhayoun et al., (1993) ActaHaematol. 89:195-199 and Toft et al. (1994) Scand. J. Clin. Lab. Invest.54:62 disclose methods for separating TC from HC using heparinsepharose, silica gel or cellulose respectively. These methods howeversuffer from the same disadvantages as the indirect methods since theyrely on the same adsorptive materials. Also, the low plasmaconcentration of holo-TC renders these methods unsuitable forcombination with existing methods of cobalamin quantification. Thenormal range of holo-TC is 35-160 pM and values below 35 pM wouldgenerally be considered as indicative of cobalamin deficiency. Thereported analytical sensitivity of most routine methods for plasmacobalamin is about 40 pM but in practice it is often much higher,typically around 90 pM. Hence, normal plasma levels of holo-TC are belowor near the sensitivity limit of the routine methods for cobalaminquantification.

Possibly the most accurate method currently recognised for determiningTC bound cobalamin involves adsorbing TC to silica and then assaying thebound fraction for cobalamin content using either an immunoassay asdescribed for example by Kuemmerle et al. (1992) Clin. Chem. 38/10:2073-2077, or a microbiological assay, the latter apparently producingthe best results. This method requires an entire working day to performonly twenty assays. It is very expensive and impractical and poorlysuited to routine clinical diagnostic laboratory investigations.

In WO 00/17659, an alternative method for determining holo-TCconcentration in which, for example, a cell-free sample is contactedwith an antibody specific for TC immobilized on magnetizable particlesand with a non-overlapping, non-immobilized, radiolabelled antibodyspecific for holo-TC. The particles are separated out using a magneticfield and washed and their radioactivity is measured so providing ameasurement of the holo-TC content of the sample. This method howeverrequires calibration and is more suited for performance in a clinicallaboratory than by a physician or physician's assistant at the point ofcare for the patient.

There thus still exists a need for an assay for holo-TC which is simpleand operable at the point of care.

We have recognized that such a simple assay can be achieved by measuringnot holo-TC content but TC saturation, i.e. the proportion of TC that isin the holo form, by using two labelled specific binding partners, onefor holo-TC and the other for apo-TC or for both holo and apo-TC and bydetermining the ratio of the signals from the two labels. Moreover suchan assay is volume independent, i.e. the quantity of the liquid sample(e.g. body fluid) used need not be precisely determined. This is a majoradvantage for point-of-care use.

Thus viewed from one aspect the invention provides an assay method fordetermining transcobalamin saturation wherein a transcobalamincontaining liquid sample is contacted with a porous substrate withimmobilized thereon a transcobalamin immobilizing ligand and with areporter-labelled transcobalamin binding partner and wherein signalsfrom reporter labels which become immobilized on said substrate aredetected, characterised in that one of said ligand or said bindingpartner comprises a first ligand or binding partner capable of specificbinding to holo transcobalamin and a second ligand or binding partnercapable of binding to apo transcobalamin or to holo and apotranscobalamin.

In one embodiment, the assay method of the invention comprises:

-   -   (i) contacting a transcobalamin containing liquid sample with a        porous substrate having immobilized thereon a        transcobalamin-immobilizing ligand;    -   (ii) before, during or after step (i) above, contacting        transcobalamin of said sample with a first and a second        reporter-labelled binding partner for transcobalamin, said first        binding partner being capable of binding both holo and apo        transcobalamin or being a specific binding partner for apo        transcobalamin and said second binding partner being a specific        binding partner for holo transcobalamin; and    -   (iii) detecting signals from the reporter labels of said binding        partners immobilized on said substrate and determining therefrom        an indication of transcobalamin saturation in said sample.

Alternatively, the substrate may have a first region having immobilizedthereon a ligand which is a specific binding partner for holotranscobalamin and a second region having immobilized thereon a ligandwhich is a specific binding partner for apo transcobalamin or which iscapable of immobilizing both holo- and apo transcobalamin and step (iii)may comprise detecting signals from said first and second regions.

By a specific binding partner or ligand is meant one which binds to TC(ie. apo-TC and/or holo-TC as required) by virtue of its specificchemical structure or conformation and not simply by virtue of anoverall physico-chemical property (such as lipophilicity) which may becommon to many components of a body fluid sample.

The specific binding partner or ligand will generally be either anantibody, an antibody fragment, a single chain antibody, an antibodyfragment dimer, trimer or tetramer, or a compound with an affinity forapo-TC and/or holo-TC, such as a cell surface receptor, a polypeptide,an oligopeptide, an oligonucleotide, a small organic chemical, etc.Other specific binding partners or ligands may be selected from acombinatorial chemistry or phage display library or specific bindingsequences of DNA or RNA.

If the specific binding partner or ligand is an antibody it may bepolyclonal but will preferably be monoclonal. Monoclonal antibodies canbe generated with much greater specificity and uniformity thanpolyclonal antibodies and this reduces cross-reactivity with othercomponents of the body fluid, in particular HC and where appropriate,the alternative conformation, e.g. the apo form of the target analyte.The uniformity and reproducibility offered by monoclonal antibodiesrelative to polyclonal antibodies ensures a greater accuracy which isvital for an assay wherein the analyte is in such low concentration.Alternatively, it may be an antibody fragment for example F(ab), F(ab′)₂or F(v) fragment. The antibodies or antibody fragments may be monovalentor divalent and may be produced by hybridoma technology or be ofsynthetic origin, and generated by recombinant DNA technology orchemical synthesis. Single chain antibodies or other antibodyderivatives or mimics could for example be used. The antibody may bedirected or raised against any epitope, component or structure of theapo and/or holo-TC protein as appropriate.

The specific binding partner or ligand (sbp) for holo-TC used in theassay method of the invention should have a specificity for holo-TC (asopposed to apo-TC) which is at least 10-fold, preferably at least50-fold, more preferably at least 100-fold, i.e. in an excess of apo atleast 10 times as much of the holo-TC sbp should bind to holo-TC than toapo-TC. In general therefore it will be preferred to select the holo-TCsbp using in vitro methods, rather than by antibody generation in a hostanimal. Accordingly screening for candidate holo-TC sbps will preferablybe done using in vitro libraries, e.g. phage display antibody(especially single chain antibody) libraries or oligonucleotide orchemical libraries. The affinity of the holo-TC sbp for holo-TC willmoreover preferably be such that nanomolar concentrations of holo-TC canbe detected.

Candidate holo-TC sbps can for example be selected by immobilizingholo-TC on a substrate (e.g. beads or sheets), e.g. by amide coupling,and panning the library in the presence of an excess of non-immobilizedapo-TC. The substrate should then be washed thoroughly and holo-TCbinding candidates should then be released and identified by the meansconventional for the library type.

Candidates may then be tested for cross-reactivity for apo-TC andcandidates having sufficient preference for holo-TC rather than apo-TCcan then be selected. Desirably candidate holo-TC sbps will also bescreened to deselect sbps which are cross-reactive for HC.

Most preferably, the binding regions of the holo-TC sbps should benon-peptidic, e.g. they may be small organic molecules oroligonucleotides (e.g. 20 to 50-mers).

Candidate apo-TC sbps can be selected in equivalent fashion; however itwill generally be preferred to use a holo-TC sbp and a non-overlappingTC sbp (i.e. one which binds to both apo and holo TC) and TC sbps arewell known from the literature (see for example Quadros et al., Biochem.Biophys. Res. Commun. 222: 149-154 (1996) and McLean et al., Blood 89:235-242 (1997)).

Once candidate holo TC sbps and TC or apo-TC sbps have been selected,they may be reporter-labelled by conventional means.

The reporters may be the same if different ligands are immobilized ondifferent regions of the substrate; however otherwise the reporters usedshould be different, i.e. their signals should be interdistinguishable.

The reporter moieties in the sbps may be any of the conventionalreporters, e.g. radiation emitters or absorbers, in particularelectromagnetic radiation emitters or absorbers, enzymes, or, lesspreferably, radiolabels, etc. In the case of enzymatic reportermoieties, the signals detected will be signals, e.g. emitted light, fromthe reaction catalysed by the enzyme. Thus the detected signals may begenerated directly or indirectly by the reporter moieties.

By means of example only, some suitable examples of coloured orfluorescent compounds which may be used to label a sbp detectably in thepresent invention are anthraquinones, azo dyes, azine dyes such asoxazines and thiazines, triazines, naturally occurring pigments such asporphyrins, phycobiliproteins, including phycoerythins and phycoquanins,chlorophylls and their analogues and derivatives, carotenoids,acrinidines, xanthenes including fluoresceins and rhodamines,indigo-dyes, thioxanthenes, coumarins, polymethines including di and triarylmethines and derivatives thereof of phthalocyanines and metalphthalocyanines.

Similarly, a wide range of radioactive compounds may be used as thesignal forming label, among them Iodine-125-labelled compounds.

Alternatively, the sbp may be conjugated to natural or syntheticcompounds which can produce a chemiluminescent signal which may beassayed in known manner (Cormier, M. J. et al.; Chemiluminescence andBioluminescence, Plenum Press, New York 1973). Suitable chemiluminescentcompounds include luciferin, oxalic esters, 1,2-dioxethane, luminol orderivatives thereof, but are not limited to these. If appropriate,hydrogen peroxide, enzymes e.g. luciferase, alkaline phosphatase orother chemicals may be used to produce the chemiluminescent signal fromthe signal-producing molecules used.

Preferably, the sbp is conjugated to a polymeric “scaffold” whichcarries a plurality of reporter moieties, e.g. enzymes, chromophores,fluorophores, etc. In this way the signal may be magnified, e.g. severalhundred-fold. Typical polymer scaffolds include dextran (e.g. Amdex),polyethylene glycol and dendrimers (e.g. starburst dendrimers).

Strongly-anionic signal forming molecules may not be preferred for usein the method of the invention, since they have a tendency to bind toserum proteins such as human serum albumin (HSA) which may be present inthe sample. Particularly suitable examples which may be used arefluorescein isothiocyanate, Rhodamine B orN-(resorufin-4-carbonyl)piperidine-4-carboxylicacid-N-hydroxysuccinimide-ester) (resos).

Where the reporter is a light emitter or absorber, it is particularlypreferred (especially for the labelled holo-TC sbp) that the reportercomprise a plurality of chromophores or fluorophores so that the signalis magnified. One particularly preferred group of reporters is the groupof reporters capable of time-delayed fluorescence, another is the groupcapable of provoking luminescence. Specific examples of reportersinclude β-galactosidase and chicken alkaline phosphatase.

It is well known in the art to immobilise affinity molecules, e.g.antibodies and antibody fragments for separation purposes, for exampleby binding or coupling the ligands, optionally by means of a linker, toany of the well known solid supports or matrices which are currentlywidely used or proposed for separation or immobilisation, and any knownmethod in the art could be used. Such solid phases may take the form ofparticles, sheets, gels, filters, membranes, fibres or capillaries.Techniques for binding the ligand to the porous substrate are thusextremely well known and widely described in the literature.

In the method of the invention, the substrate is preferably a poroussheet or strip (e.g. of a cellulosic material, preferablynitrocellulose), especially preferably mounted on a non-porous support,optionally with an absorbent backing which serves to promote absorptionof the liquid sample into the substrate by capillary action. Thus in onepreferred embodiment the substrate layer, preferably backed by anabsorbent layer, is mounted on a dipstick. In another preferredembodiment the substrate layer preferably backed by an absorbent layer,is held in a plastic casing provided with an aperture for application ofthe liquid sample to the exposed surface of the substrate layer and forreading the signals from the substrate layer.

Signal generation, if required, may be by whatever means are suitablefor the selected labels, e.g. by exposure to light or to a substrate foran enzymatic label, and signal detection may be by any detector suitablefor detecting the emitted signals, e.g. radioactivity or lightdetectors. Especially preferably, where the signals are light signals, adigital camera will be used as the detector, if necessary provided witha filter, prism or other means to ensure that the desired wavelengthband is allowed to reach the detector.

Detection of the signals from the two labelled sbps may be simultaneousor sequential. Simultaneous detection is preferred.

Detected signal manipulation to generate a quantitative,semi-quantitative or qualitative indication of TC saturation isconveniently performed by a computer, preferably one built into theapparatus used to perform the assay and arranged to control performanceof the assay. Where the signal forming labels are different in the sbps,the assay will require calibration in conventional fashion to transformthe signal ratio (Sa/Sb or Sa/(Sa+Sb) where Sa is the holo-TC sbp signaland Sb is the TC or apo-TC sbp signal respectively) into a TC saturationvalue. Accordingly the assay may if desired be provided with calibrationstandards containing apo and holo-TC in known ratios.

In an alternative embodiment, any method of the current invention may becarried out on the surface of a chip-type substrate within a surfaceplasmon resonance (SPR) detector. In such a method, the need for activelabelling of the specific ligands or substrates is avoided and bindingis detected directly by the additional mass of the specific bindingligands or specific binding partners binding to the surface. In apreferred example of this alternative, a TCII binder is immobilised onthe surface of an SPR “chip” (e.g. by gold-coating of the chip, followedby absorption of a thiol-conjugated TCII binder or by amide coupling ofthe binder to a dextran-coated chip). The chip is then placed in thesurface plasmon resonance detector and a solution containing the sampleis caused to flow over the chip, so binding holo-TC and apo-TC from thesample onto the immobilised ligand. A solution containing a firstspecific binder with specificity for apo-TC or for both apo-TC andholo-TC is then caused to flow over the chip and the binding measured bysurface plasmon resonance to give a first signal. This first specificbinder is then optionally washed off and a second specific binder withspecificity for holo-TC only is passed over the chip. A second signal,representing the binding to holo-TC is detected by surface plasmonresonance. This difference between the first and second SPR is then usedto calculate the TCII saturation. By using SPR, any ligands or specificbinding partners described as “labelled” or “reporter labelled” may bedetected by mass without any additional or active label. In this casethe “label” is simply the mass of the specific binding partner orligand.

The sample applied to the substrate is preferably a body fluid ortissue-derived liquid, preferably a cell-free liquid, especially plasmaor serum from a mammalian, especially human, subject.

Viewed from a further aspect the invention provides an assay kitcomprising:

a porous substrate having immobilized thereon a TC-immobilizing ligand;

a first and a second reporter-labelled binding partner fortranscobalamin, said first binding partner being capable of binding bothholo and apo transcobalamin or being a specific binding partner for apotranscobalamin and said second binding partner being a specific bindingpartner for holo transcobalamin;

optionally, a washing agent;

optionally, a substrate for an enzymatic reporter label;

optionally, at least one calibration standard containing apo and holo-TCat known ratio; and

optionally, a detector capable of detecting signals from said reporterlabels.

In an alternative format, the transcobalamin saturation can bedetermined by binding to different regions of the substrate two ligands,a first which immobilizes holo-TC and a second which immobilizes apo-TC(or apo and holo-TC), and contacting the substrate with theTC-containing sample and a reporter labelled sbp for TC. In this form,which forms a further aspect of the invention, the signals are read fromthe different regions of the substrate and the signal ratio Sa/(Sa+Sb)or Sa/Sb (where Sa is the signal for the holo-TC ligand region and Sb isthe signal for the apo-TC or holo and apo-TC ligand region) isindicative of the TC saturation level. The “regions” of the substrate inthis or other aspects of the invention may for example be differentareas of the surface of a membrane, different membrane surfaces on adipstick, different membranes in a casing, or different sets ofseparable beads (e.g. one set being magnetizable, the other not).

In this format, the assay method of the invention typically comprises:

-   -   (i) contacting a transcobalamin containing liquid sample with a        porous substrate having immobilized thereon in different regions        thereof a first and a second transcobalamin-immobilizing ligand,        said first ligand being capable of specific binding to holo        transcobalamin and said second ligand being capable of        immobilizing apo or apo and holo transcobalamin;    -   (ii) before, during or after step (i), contacting transcobalamin        of said sample with a reporter-labelled binding partner for        transcobalamin; and    -   (iii) detecting signals from the reporter labels of said binding        partner immobilized on the different regions of said substrate        and determining therefrom an indication of transcobalamin        saturation in said sample.

Viewed from a yet further aspect the invention thus provides an assaykit comprising:

a porous substrate having immobilized thereon in different regionsthereof a first and a second transcobalamin-immobilizing ligand, saidfirst ligand being capable of specific binding to holo transcobalaminand said second ligand being capable of immobilizing apo or apo and holotranscobalamin;

a reporter-labelled binding partner for transcobalamin;

optionally, a washing agent;

optionally, a substrate for an enzymatic reporter label;

optionally, at least one calibration standard containing apo and holo-TCat known ratio; and

optionally, a detector capable of detecting signals from said reporterlabels.

The invention will now be described further with reference to thefollowing non-limiting Examples and to the drawings in which;

FIG. 1 represents a substrate upon which TC ligands have beenimmobilised;

FIG. 2 represents a substrate having two regions, one containingimmobilised ligands specific to holo-TCII and a second havingimmobilised ligands specific to apo-TCII; and

FIG. 3 represents a chip within a surface plasmon resonance detectorhaving antibodies for TC immobilised on the surface.

In FIG. 1, “label-spb1” represents a first specific binding partnerhaving a first label attached and “sbp-label2” represents a secondspecific binding partner having a second label attached. It can be seenthat the signal from label1 corresponds to the holo-TC level and thesignal from label2 corresponds to the total apo- and holo-TC levels.

In FIG. 2, it can be seen that holo-TCII has bound to the first (lefthand) region and apo-TCII to the second (right hand) region. The“sbp-label” represents a specific binding partner for TC having anattached label. In this way, the signals from the two regions representthe holoTC and apoTC content respectively.

In FIG. 3, a chip surface has been modified by attachment of a firstmonoclonal antibody (mAb1). This binds to apoTC and to holoTC from asample. The second antibody (mAb2) also binds either holo-TC or apo-TC,thus giving an SPR signal for total TC content when this is added. Thethird antibody (mAb3) binds only to holo-TC and so gives an SPR signalrepresenting the holo-TC content.

EXAMPLE 1 Generation of holo-TC sbp

A) Immobilization of holo-TC on Beads

One milliliter of 0.2M 1-ethyl-3-(3-dimethylamino-propyl)-carbodiimide(EDAC) in 0.1M 2-(N-morpholine)-ethanesulfonic acid (MES), pH 5.0 ismixed with 1.0 mL 2% (w/v) carboxylate-modified beads (1 μm diameterMerck-Estapor) in 0.1M MES, pH 5.0. The mixture is treated with bathsonication to disperse the reagents and is then rotated end-over-end for1 h at room temperature. The mixture is centrifuged at 300 g and thepellet is washed with 0.1 M MES, pH 7.0, centrifuged and is resuspendedin 1.0 mL deionized water. The EDAC activated beads are mixed in a smallplastic vial with the same volume of 1 mg/mL holo-TC in 0.1M3-[N-morpholino]-propane sulphonic acid (MOPS), pH 7.5 and rotatedovernight at room temperature. Thereafter, the mixture is washed twicewith 0.05% Tween 20 (aq) and twice with 50 mM Tris buffer, pH 7.4,containing 5 mg/mL BSA. The coated beads having a monolayer of holo-TCof about 10 μg/mg are stored at 0.1% in 50 mM Tris, pH 7.4, 0.15M NaCl,and 1 mg/mL BSA.

B) (i) Biopanning of Aptamer Libraries with holo-TC as the TargetMolecule

A library of double-stranded DNA sequences is prepared by a mixture ofchemical and enzymatic steps. Typically, 10¹⁴-10¹⁵ single stranded DNA(ssDNA) sequences of length 40 nucleotides are made synthetically andare then converted to the double-stranded form by enzymatic means priorto PCR amplification. Second, the double-stranded DNA is transcribed toyield a library of single-stranded RNA or modified RNA. Third, the RNAlibrary is challenged with the intended target molecule. The selectedmolecules are reverse transcribed and then amplified by PCR (for DNAlibraries PCR is sufficient). The subset of sequences that bind to thetarget becomes the pool for a second round of biopanning. Usually theselection process takes from 7 to 15 rounds.

Five nmol of gel-purified, synthetic template DNA containing 40nucleotides contiguous sequence flanked by defined primer annealingsequences SEQ ID NO. 1 [5′-GGGAGGACGATGCGG-(N)₄₀-CAGACGACTCGCCCGA-3′] isamplified by four PCR cycles with primers SEQ ID NO. 25′-TAATACGACTCACTATAGGGAGGACGATGCGG-3′ and SEQ ID NO. 35′-TCGGGCGAGTCGTCTG-3′. Approximately 800 pmol of the PCR-derivedtemplate DNA (about 5×10¹⁴ molecules) are transcribed in vitro by T7 RNApolymerase (1000 U) in a 3 mL transcription reaction consisting of 40 mMTrisHCl (pH 8.0), 12 mM MgCl₂, 1 mM Spermidine, 5 mM dithiothreitol(DTT), 0.002% Triton X-100 (v/v), 4% polyethylene glycol (w/v) and 2 mMeach of ATP, guanidine-5′-triphosphate (GTP), 2′—NH₂CTP and 2′—NH₂UTP(CTP=cytidine 5′-triphosphate and UTP=uridine 5′-triphosphate). Fulllength transcription products are purified on 8% polyacrylamide gelsunder denaturing conditions, suspended in binding buffer (10 mM TrisHCl,0.1 mM EDTA, 2.5 mM MgCl₂, pH 6.8), heated to 70° C. and chilled on ice.

The RNA pool is incubated at 37° C. for 15 min with about 10 μg holo-TCimmobilized on beads and 100 μg of apo-TC free in the solution ascompetitor. The beads are separated by means of centrifugation and areimmediately washed with 5 mL binding buffer. Bound RNA molecules areeluted from the beads by lowering the pH, recovered by ethanolprecipitation and reverse transcribed by avian myeloblastosis virustranscriptase (Life Sciences) at 48° C. for 45 min with the DNA sequenceSEQ ID NO. 3 5′-TCGGGCGAGTCGTCTG-3′ as the primer. Following PCRamplification of the cDNA, the resulting duplex DNA template istranscribed in vitro to obtain RNA for the next round of selection. Theamount of holo-TC coated beads in the binding reaction are successivelyreduced to progressively increase the selection pressure. The selectionprocess is repeated until the affinity of the enriched RNA pool forholo-TC is substantially increased. At this point, cDNA is amplified byPCR with primers SEQ ID NO. 45′-CCGAAGCTTAATACGACTCACTATAGGGAGGACGATGCGG-3′ and SEQ ID NO. 55′-GCCGGATCCTCGGGCGAGTCGTCTG-3′, which introduces BamHI and HindIIIrestriction sites (underlined) at the 5′ and 3′-ends of the PCRproducts, respectively. The PCR products are digested with BamHI andHindIII and cloned into pUC18 that has been digested with the sameenzymes and introduced into Escherichia coli SURE (Stratagene) byelectroporation. Plasmids are isolated from single bacterial clones andare screened and sequenced using standard techniques as described ine.g. Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual, 2ndedition Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.,pp C1.

In order to confer RNA nuclease resistance, the RNA molecules may bemodified at the 2′ position of the sugar, e.g. amino substitution.

The biopanning protocol will be essentially identical whether thelibrary is based on RNA aptamers or ssDNA aptamers, or whether thelibrary is naive or biased. Basically, the difference will be that ssDNAlibraries will not require transcription.

(ii) Biopanning of Phage Display Peptide or Antibody Libraries withholo-TC

One pmol of phage display library is mixed with 10 μg of holo-TCimmobilized on beads and 100 μg of apo-TC in 100 μL PBS-Tween with 1mg/mL BSA or 3% BLOTTO. (BLOTTO is 5% w/v non-fat powdered milk in 100mM sodium phosphate, 150 mM sodium chloride, pH 7.4, 0.01% Antifoam A,0.01% thimerosal). The mixture is incubated at 4° C. overnight and thebeads are separated through centrifugation and washed 9 times with 1 mLPBS-Tween and one time with normal saline to remove any bufferingcapacity. The beads are resuspended in 600 μL of 0.1 M glycine-HClbuffer, pH 2.2 and after 15 min the beads are separated throughcentrifugation at 3000 g for 3 mm. The supernatant is removed andneutralized with 36 μL of 2M Tris, pH 9.0, and mixed with 400 μL ofEscherichia coli (e.g. K91-Kan). Plasmids are isolated from singlebacterial clones and are screened and sequenced using standardtechniques as described in e.g. Sambrook J. et al., supra. The bindingand elution reactions are performed at least three times. BSA and BLOTTOare used alternating in the binding reaction to prevent enrichment ofphages binding to these proteins.

EXAMPLE 2 Immobilization of TC Specific Binder on Cellulose Paper

Cellulose paper (e.g. Whatman No. 52) is activated by first swelling itin deionized water for 3 min and then treating it with 3% CNBr (aq). ThepH is raised to 10.5 by addition of 1 mM NaOH. After 30 min the paper iswashed with 12×500 mL 5 μM NaHCO₃, 2×500 mL deionized water, 2×500 mL ofeach of 25%, 50%, and 75% acetone. Finally it is washed with 4×500 mLacetone and air dried at room temperature.

Covalent coupling of anti-TC antibody is performed by dilution of theantibody to 10 μg/mL in 100 mL 0.1M NaHCO₃ and adding 20 g of cut paper.The mixture is stirred gently for 3 h at 4° C., then washed at roomtemperature for 10 min with 2×100 mL 0.5 mM NaHCO₃, for 3 h with 100 mL50 mM ethanolamine in 0.1 M NaHCO₃, for 10 min with 2×100 mL 0.5 mMNaHCO₃, for 30 min with 100 mL 0.1 M sodium acetate, pH 4.0, and twicewith 50 mM phosphate buffer, pH 7.5, containing 0.15M NaCl, 0.1% Tween20, and 0.1% gelatin. The antibody loaded paper is stored in a smallvolume of this buffer.

Sections of the antibody loaded paper are then adhered to a flexibleplastic dipstick.

EXAMPLE 3 Assay

50 μL of human serum is applied to the membrane pad of a dipstickprepared according to Example 2. After 5 min, the dipstick is rinsedwith 100 μL of Tris-buffered saline (TBS-100 mM Tris, pH 7.2, 150 mMsodium chloride) and 50 μL of a Detection solution is added andincubated for 5 minutes. The Detection solution contains anon-overlapping, anti-human TC specific monoclonal antibody conjugatedto β-galactosidase and a holo-TC sbp according to Example 1 (or anon-overlapping anti-human holo-TC monoclonal antibody) conjugated tochicken alkaline phosphatase. The dipstick is then rinsed with 100 μLTBS. 50 μL of a luminescent substrate to alkaline phosphatase in TBS isadded (e.g. 0.25 mM CPD-Star from Tropix). The dipstick is measured in aluminometer for 1 s to 15 minutes. The dipstick is then rinsed with 100μL PBS (phosphate buffered saline), and 50 μL of a luminescent substratefor β-galactosidase in PBS (e.g. 0.1 mM Galacton-Star from Tropix), andan alkaline phosphatase inhibitor (e.g. 40 μMcyclohexane-trismethylenesulfonate), are added. The dipstick is thenread once more in the luminometer. Whereas the first reading gives theholo-TC content on the dipstick, the second reading gives the total TCcontent. The ratio of the two gives the volume independent value for thecobalamin saturation of TC in the sample.

EXAMPLE 4 Development of Mouse Monoclonal Antibodies Specific for HumanTranscobalamin and Human holo-transcobalamin i) Immunization.

BALB/c female mice were immunized i.p. with 20 μg of recombinant humanholo-transcobalamin mixed with AdjuPrime Immune modulator (Pierce, Ill.,USA), followed by two booster injections of 20 μg at four weekintervals.

ii) Fusion.

Four days after the final boost, spleens were removed and splencystesfused to the HAT (Hypoxanthine, Aminopterin, Thymidine; Sigma) sensitiveplasmacytoma OUR1 (a sub-clone of X63-Ag8.653) using PEG (BoehringerMannheim, Germany). Fusion products were plated over 5×96F trays (Nunc)in the presence of HAT in culture medium (DMEM/Ham's F12 (Invitrogen)plus 10% CPSR3 (Sigma). Fusions were fed after 1 week with culturemedium containing HT (Sigma).

iii) Primary screening.

After two weeks, medium from the hybridomas was screened using a solidphase capture assay. The cell media were mixed with 10 μL of 1% (w/v)suspension of 1 μm magnetizable beads coated with a sheep anti-mouse IgGantibody (Merck-Estapor, France) and kept at ambient temperature for 1h. The magnetizable beads with bound mouse monoclonal antibodies wereisolated by using a magnet, and washed four times with 1 mL phosphatebuffer, pH 7.2, 0.15M NaCl and 1 mg/mL bovine serum albumin. Washedbeads were resuspended in 100 μL of pooled human serum (Scantibodies,USA) which had been pretreated with 57Co-labeled cobalamin (ICN, USA) toconvert apo-transcobalamin in the serum into 57Co-labeled holoTC. Themixtures were kept at ambient temperature for 30 min and beads isolatedby using a magnet. The radioactivity associated with the beads wascounted in a gamma counter.

iv) Cloning.

Wells positive for anti-transcobalamin antibodies were cloned bylimiting dilution over 96wellF trays (Nunc), pre-seeded with Balb/cperitoneal feeder cells (10,000 per well). Positive clones wereselected, and recloned until 100% of the subclones were producingspecific antibody. Cell stocks were frozen in liquid nitrogen, in CPSR3(Sigma) containing 10% DMSO (Sigma).

Secondary screening. Antibodies from cell media were isolated onmagnetizable beads as described above. Ten μL of the antibody coatedbeads were mixed with serum prelabeled with 57-Co, as described above,in the presence of increasing concentrations of recombinant, humanapo-transcobalamin or recombinant, human holo-transcobalamin.

Two anti-transcobalamin antibodies (mAb1 and mAb2) were selected basedon their affinity for both apo- and holo-transcobalamin and onemonoclonal antibody (mAb3) was selected based on specificity forholo-transcobalamin. The respective clones were expanded in vitro inTecnomouse, CL1000. Antibodies were purified from cell medium by proteinA affinity chromatography. In short, the antibody containing culturesupernatant was diluted 1:1 in 0.1M Tris, pH 8.0, containing 1M NaCl(binding buffer) and applied to the protein A column, which had beenpre-equilibrated with binding buffer. The protein A column was thenextensively washed with 15 column volumes of binding buffer. Theantibody was eluted with 0.1M citrate buffer, pH 4.0, neutralized to pH7.0 with 1M Tris buffer, pH 8, and buffer changed on a Sephadex G-25column (Pharmacia) to 0.1M phosphate, pH 7.2, containing 0.15M NaCl.

EXAMPLE 5 Immobilization of TC Specific Monoclonal Antibody mAb1 onDextran Coated Surface

The carboxylated surface of the dextran coated chip (Pharmacia, Sweden)was activated with 0.05M N-hydroxysuccinimide (NHS)/0.2MN-ethyl-N′-[3-(dimethylamino)propyl]carbodiimide hydrochloride (EDC),rinsed with deionized water and coated covalently with mAb1 (fromExample 4) at 50 μg/mL in 0.01 HEPES buffer, pH 7.4, containing 0.15MNaCl, 0.003M EDTA, and 0.005% polysorbate (HBS). Unreacted NHS wasblocked by 1M ethanolamine and the chip washed extensively with HBS.

EXAMPLE 6 Binding of holo-transcobalamin, mAb2, and mAb3 to ImmobilizedmAb1

The binding events were followed in real time using surface plasmonresonance and performed on a Biocore instrument (Biacore, Sweden). Theamount of free ligand binding to the chip was measured in response units(RU), which reflect both the mass of ligand binding and its affinity forthe immobilized target. The chip with immobilized mAb1 (Example 5) wasintroduced into the instrument, after which was injected 5 μL of 100 nMholo-transcobalamin, 5 μL of 50 μg/mL mAb2, and 5 μL of 50 μg/mL mAb3successively with washing steps between each. After flowingholo-transcobalamin through the instrument, 565 RU of mAb3 bound to theimmobilised mAb1. In the absence of holo-transcobalamin neither mAb2 normAb3 bound to the chip with the immobilized mAb1.

1. An assay method for the determination of transcobalamin saturation ina transcobalamin containing liquid sample, as a ratio ofholo-transcobalamin to total transcobalamin content, said methodcomprising: (i) contacting said sample with a porous substrate havingimmobilized thereon in a first region a firsttranscobalamin-immobilizing ligand, being capable of specific binding toholo transcobalamin, whereby to generate a first transcobalamin boundregion having holo transcobalamin bound thereto, and in a second regiona second transcobalamin-immobilizing ligand being capable ofimmobilizing apo or apo and holo transcobalamin, whereby to generate asecond transcobalamin bound region having apo or apo and holotranscobalamin bound thereto; (ii) before, during or after step (i)above, contacting transcobalamin of said sample with a reporter-labeledbinding partner for transcobalamin, whereby to immobilize saidreporter-labeled binding partner by binding to said first and secondtranscobalamin bound regions; and (iii) detecting signals from thereporter-labeled binding partner immobilized on the first and secondregions, determining therefrom a holo transcobalamin level and a totaltranscobalamin level in said sample and calculating the transcobalaminsaturation in said sample as the ratio of the holo transcobalamin levelto the total transcobalamin level.
 2. The method of claim 1 wherein saidligand capable of specific binding to holo transcobalamin comprises anon-peptidic holo transcobalamin binding moiety.
 3. The method of claim1 wherein said ligand capable of specific binding to holo transcobalamincomprises a monoclonal antibody.
 4. The method of claim 1 wherein saidligand capable of specific binding to holo transcobalamin has aspecificity for holo transcobalamin vs apo transcobalamin which is atleast 10-fold.
 5. The method of claim 1 wherein said signals areelectromagnetic radiation.
 6. The method of claim 5 wherein said signalsare detected by a digital camera.
 7. The method of claim 1 wherein saidsample is blood or a blood-derived liquid.
 8. The method of claim 1wherein said substrate is a cellulose sheet.
 9. The method of claim 1wherein said substrate is mounted on a dipstick.
 10. The method of claim1 wherein in step (i) said sample is applied to one face of said poroussubstrate and is drawn into said porous substrate by capillary flowpromoted by a water absorbent material adjacent the opposed face of saidporous substrate.
 11. The method of claim 1 wherein said reporterlabelled specific binding partner is labelled only by its own mass andis detected by surface plasmon resonance.
 12. An assay kit comprising: aporous substrate having immobilized thereon in different regions thereofa first and a second transcobalamin-immobilizing ligand, said firstligand being capable of specific binding to holo transcobalamin and saidsecond ligand being capable of immobilizing apo or apo and holotranscobalamin and; a reporter-labelled binding partner fortranscobalamin.
 13. The kit of claim 12 additionally comprising awashing agent.
 14. The kit of claim 12 additionally comprising at leastone substrate for an enzymatic reporter label
 15. The kit of claim 12additionally comprising at least one calibration standard containing apoand holo-TC at known ratio.
 16. The kit of claim 12 additionallycomprising a detector capable of detecting signals from said reporterlabels.